Th1-type and Th2-type immune responses were originally defined as immune responses mediated by two distinct CD4+ T cell (helper T cell—Th) subsets that secrete two different groups of cytokines. For a recent review, see Berkers and Ovaa, Trends Pharmacol. Sci., 2005, 26(5):252-257, and references cited therein.
Th1 cells secrete Th1-type cytokines including interferon-gamma (IFN-γ) and interleukin 12 (IL-12). The principal function of Th1-type cytokines is to support cell-mediated immunity that results in the elimination of tumor cells, viruses and other intracellular pathogens by stimulating phagocyte-mediated defense and increasing the activity of CD8+ T cells (cytotoxic T cells) and natural killer (NK) cells. In addition, Th1 cytokines inhibit the switching of immunoglobulin synthesis by B cells, and suppress the production of certain immunoglobulin isotypes such as IgG1 and IgE, the latter being particularly important for causing allergies. IL-12 is secreted mainly by antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages, and activates CD8+ T cells and NK cells. Th2 cells secrete Th2-type cytokines including IL-4, IL-5, IL-10, and IL-13. The principal function of Th2-type cytokines is to support humoral immunity (e.g., stimulate IgE and eosinophil/mast cell-mediated immune reactions) and to down-regulate Th1-type immune responses.
Dysregulation of the balance between Th1- and Th2-type immune responses causes disease. Many types of cancer are characterized by a predominant Th2-type response, and many pathogens evade the immune system by producing cytokines that shift the Th1-Th2 balance to the Th2 mode (Wilson and Delovitch, 2003, Nat. Rev. Immunol., 3: 211-222; Dredge, Cancer Immunol. Immunother., 2002, 51:521-531; Servet and Zitvogel, Curr Mol. Med., 2002, 2:739-756; Pinto, Pediatrics, 2006, Apr. 17 [Epub ahead of print]). Many autoimmune diseases such as asthma are also characterized by the shift of the Th1-Th2 balance to the Th2 mode. On the contrary, autoimmune diseases such as type 1 diabetes and multiple sclerosis are mediated by autopathogenic Th1 cells and are characterized by hyporesponsive Th2 cells, which leads to a Th1-like cytokine profile (Hayakawa et al., 2004, Curr. Med. Chem., 11: 241-252; Wilson and Delovitch, 2003, Nat. Rev. Immunol., 3: 211-222; Van Kaer, 2004, Immunol. Cell Biol., 82: 315-322).
Natural killer T (NKT) cells have a crucial role in regulating Th1- and Th2-type immune responses. NKT cells are a unique population of lymphocytes that co-express markers of NK cells along with a semi-invariant T cell receptor (TCR). In mice, the TCR of most NKT cells consists of an invariant Vα chain encoded by the Vα14 and Jα18 gene segments paired with a variable set of Vβ chains encoded mainly by the Vβ8.2, Vβ7 or Vβ2 gene segments. This TCR enables NKT cells to recognize the major histocompatibility complex (MHC) class I-like molecule CD1d, which is capable of presenting hydrophobic molecules such as lipids and hydrophobic peptides to NKT cells.
Thus far, only a few molecules have been shown to activate NKT cells. Of these, alpha-galactosylceramide (α-GalCer), a glycolipid originally extracted from Okinawan marine sponges (Natori et al., Tetrahedron, 50: 2771-2784, 1994) is the best characterized. A synthetic analog of α-GalCer, KRN 7000 (2S,3S,4R)-1-O-(α-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-,-octadecanetriol, can be obtained from Pharmaceutical Research Laboratories, Kirin Brewery (Gumna, Japan) or synthesized as described in Morita et al., J. Med. Chem., 1995, 38: 2176-2187. Other α-GalCer derivatives are described in U.S. Pat. No. 5,780,441 (Kirin). Following the initial disclosures by Kirin, α-GalCer has shown potential in the treatment of several diseases, including primary tumors and their metastases, infectious diseases such as malaria and hepatitis B, and several autoimmune diseases such as diabetes and asthma (see Hayakawa et al, 2004, Curr. Med. Chem., 11:241-252; Wilson and Delovitch, 2003, Nat. Rev. Immunol., 3:211-222; Taniguchi et al, 2003, Annu. Rev. Immunol., 21:483-513; Van Kaer 2004, Immunol. Cell Biol. 82:315-322). It has also been demonstrated that α-GalCer can be used as an adjuvant capable of enhancing and/or extending the duration of the protective immune responses induced by other antigens (see US 2003-0157135 and Gonzalez-Aseguinolaza et al., J Exp Med., 2002, 195:617-24).
α-GalCer can activate NKT cells both in vitro and in vivo (Kawano et al., 1997, Science, 278:1626-1629; Burdin et al., 1998, J. Immunol., 161:3271-3281; Spada et al., 1998, J. Exp. Med., 188:1529-1534; Brossay et al., 1998, J. Exp. Med. 188:1521-1528). As shown in FIG. 1, α-GalCer, when present with CD1d by APCs such as monocytes, monocyte-derived immature dendritic cells and macrophages, interacts with the TCR of NKT cells, which subsequently activate both the NKT cells and the APCs, and lead to the production of both the Th1-type cytokine IFN-γ and Th2-type cytokine IL-4 by NKT cells. The IL-12 receptor is then activated on the cell surface of the NKT cells and, simultaneously, IL-12 is produced by the activated APCs. IL-12 produced by the APCs induces a second wave of IFN-γ from the NKT cells and activates NK cells to also produce IFN-γ (Hayakawa et al, 2004, Curr. Med. Chem., 11:241-252; Kawano et al., 1997, Science, 278, 1626-1629; Godfrey et al., 2000, Immunol. Today, 21:573-583; Wilson et al., 2002, Trends Mol. Med., 8:225-231; Matsuda et al., 2000, J. Exp. Med., 192:741-753). Activation of NKT cells by α-GalCer thus may result in the secondary activation of several other cell types, including NK cells, B cells, CD8+ T cells, dendritic cells and myeloid cells and in the differentiation of CD4+ T cells into either Th1 or Th2 cells.
It has been demonstrated that the administration of α-GalCer to mice resulted rapidly in strong anti-malaria activity, inhibiting the development of intra-hepatocytic stages of the rodent malaria parasites, P. yoeli and P. berghei (Gonzalez-Aseguinolaza et al., 2000, Proc. Natl. Acad. Sci. USA, 97: 8461-8466). α-GalCer was unable to inhibit parasite development in the liver of mice lacking either IFN-γ or the IFN-γ receptor, indicating that the anti-malaria activity of the glycolipid is primarily mediated by IFN-γ. IL-4 stimulated by α-Gal-Cer allows the glycolipid to ameliorate a number of different autoimmune diseases, including autoimmune type 1 diabetes and autoimmune encephalomyelitis (Wilson et al., 2002, Trends Mol. Med., 8:225-231).
Importantly, in addition to its ability to stimulate immune responses, it has been demonstrated that α-GalCer, independently of its dosage, does not induce toxicity in rodents and monkeys (Nakagawa et al., 1998, Cancer Res., 58: 1202-1207).
The effectiveness of α-GalCer therapy, however, is severely limited by the concomitant stimulation of both Th1- and Th2-type cytokines (i.e., IFN-γ, IL-12 and IL-4) (Pal et al., 2001, J. Immunol., 166:662-668; Berkers and Ovaa, Trends Pharmacol. Sci., 2005, 26:252-257). Indeed, little effect was observed in patients with solid tumors in a Phase I study with α-GalCer (Giaccone et al., 2002, Clin. Cancer Res., 8: 3702-3709). Treatment with α-GalCer has been shown to be more effective if the cytokine profile of NKT cells is shifted, e.g., towards Th1-type by administration of CD1d-pulsed dendritic cells (Fujii et al., 2002, Nat. Immunol., 3: 867-874).
An α-GalCer analog that could selectively induce Th1- or Th2-type immune response would thus have a more promising therapeutic potential.
Several α-C-GalCer analogs have been recently developed, where a carbon atom replaces the oxygen atom of the glycosidic bond. See, e.g., U.S. Pat. No. 6,635,622; Schmieg et al, 2003, J. Exp. Med, 198(11):1631-1641; Chen et al., Org. Lett., 2004, 6:4077-80; Yang et al., Angew Chem Int Ed Engl., 2004, 43:3818-22, and commonly owned U.S. patent applications Ser. No. 10/462,211 (US 2004-0127429); Ser. No. 11/193,852 (US 2006-0019246); Ser. No. 11/096,340 (US 2005-0222048). Such analogs are resistant to deglycosylation and therefore have a longer shelf-life (Bertozzi et al., Synthesis of C-glycosides: stable mimics of O-glycosidic linkages. In Modern Methods in Carbohydrate Synthesis. Khan and O'Neill, editors. Harwood Academic Publishers, London, UK, 1996, p. 316-351; Bertozzi et al., 1992. J. Am. Chem. Soc., 114:10639-10641; Levy and Tang, The Chemistry of C-Glycosides, Elsevier Science Ltd., 1995; Postema, C-Glycoside Synthesis, CRC Press, Inc., 1995).
α-C-GalCer CRONY 101 was the first example of a C-glycoside that has a significantly improved therapeutic potential compared with its O-glycosidic counterpart. As demonstrated in Schmieg et al. (2003, J. Exp. Med., 198: 1631-1641) and Yang et al. (2004, Angew. Chem. Int. Ed. Engl., 43: 3818-3822), in vivo administration of CRONY 101 results in diminished production of the Th2-type cytokine IL-4 (as compared to α-GalCer) and enhanced, prolonged production of the Th1-type cytokines IFN-γ and IL-12 leading to a 100 and 1000-fold improved activity against melanoma metastases and malaria, respectively.
The Th1-type cytokine IL-12 has recently attracted a lot of attention because of its essential role in the interaction between the innate and adaptive arms of immunity by regulating inflammatory responses and innate resistance to infection and cancer (reviewed in Colombo and Trinchieri, Cytokine Growth Factor Rev., 2002, 13:155-68; Watford, Cytokine Growth Factor Rev., 2003, 14:361-368). Endogenous IL-12 is required for resistance to many pathogens and tumors. Indeed, in experimental tumor models, recombinant IL-12 treatment has a dramatic anti-tumor effect on transplantable tumors, chemically induced tumors, and tumors arising spontaneously in genetically modified mice.
As specified above, IL-12 is mostly secreted by various APCs such as dendritic cells and macrophages and contributes to Th1-type immune response (Roitt, Brostoff, Male, Immunology, Mosby ed., 6th ed.; Ma and Trinchieri, Adv Immunol., 2001, 79:55-92; Hilkens, Blood, 1997, 90:1920-1926; Szabo, Annu. Rev. Immunol., 2003, 21:713-58). IFN-γ and a cascade of other secondary and tertiary pro-inflammatory cytokines induced by IL-12 have a direct toxic effect on the infected and tumor cells or may activate potent anti-angiogenic mechanisms. The stimulating activity of IL-12 on antigen-specific immunity relies mostly on its ability to determine or augment Th1 and cytotoxic T lymphocyte responses. Because of this ability, IL-12 has a potent adjuvant activity in cancer and other vaccines. The promising data obtained in the pre-clinical models of anti-tumor immunotherapy have raised much hope that IL-12 could be a powerful therapeutic agent against cancer. However, excessive toxicity observed in the IL-12 clinical trials point to the necessity to achieve IL-12 activation in a local rather than systemic fashion.